Intermediate filaments (IF) comprise assemblies of subunits belonging to a superfamily of α-helical proteins that fall into one of six major classes. Typically, IF proteins have common secondary structural characteristics that can generally be described as a monomeric form containing a central α-helical domain and head and tail globular domains. The central α-helical domain in IF proteins is highly conserved, with variation coming largely from differences in primary structure in the head and tail domains. In order to form intermediate filaments, these monomeric species polymerize to form elongated macromolecular complexes with a highly ordered superstructure. Two primary IF proteins include acidic and basic keratins (Type I and Type II IF protein classes respectively). Type I and Type II keratin monomers are generally expressed in epithelial cells and neither Type I nor Type II keratin monomers are able to assemble into a keratin filament on its own. Type I and Type II keratin monomers generally associate in a 1:1 ratio to form heterodimers, which further associate to assemble into heteropolymeric keratin filaments.
Keratin IF proteins can be further described as being from the “soft” epithelial sub-family or from the “hard” trichocytic sub-family. Approximately 20 keratins, also known as cytokeratins, are from the “soft” sub-family and consist of intracellular proteins making up cytoskeletal elements in epithelial cells. About 17 keratins are known to belong to the “hard” trichocytic sub-family, and these keratins make up structural appendages such as hooves, fingernails, fur, feathers, and hair fibers.
Research has shown that the process of protein self-assembly is highly sensitive to changes in the macromolecular complexes, namely dimers and tetramers. Moreover, the mere presence of damaged fragments of proteins can interfere with the self-assembly process and/or disrupt superstructure that has already formed—such holds true for keratin proteins as well. Keratin monomers form particularly strong macromolecular complexes, especially at the dimer and tetramer level, which are not easily denatured or broken down into their monomeric units. The problem in denaturing these complexes, however, is that the chemical methods used to break apart the keratin superstructure, primarily disulfide bonds, and the subsequent protein solubilization techniques, can result in significant damage to the keratin monomers. This damage often goes undetected and is detrimental to the formation, properties, and performance of keratin biomaterials. Recombinant keratins have yet to offer any promising alternatives to the field of keratin biomaterials as recombinant keratins are currently difficult and expensive to produce. To date, the entirety of keratin biomaterials technology has been based on extracting keratins from tissues such as hair fibers, wool, feathers and the like.
Previously described keratin biomaterials are made from keratin monomers, typically in the range of 40-60 kilo Daltons (kDa) and do not disclose the use of a purified keratin nanomaterial. Rouse J G, Van Dyke M E. A review of keratin-based biomaterials for biomedical applications. Materials 2010; 3:999-1014; Van Dyke M E. Hydrogel with controllable mechanical, chemical, and biological properties and method for making same. U.S. Pat. No. 7,001,987. Feb. 21, 2006; Van Dyke M E, Saul J M, Smith T L, de Guzman R. Controlled delivery system. U.S. Pat. App. Pub. No.: 2011/0217356. Filed Mar. 7, 2011. As described herein, a purified keratin nanomaterial includes keratins essentially devoid of structural damage and defects as well as damaged proteins and peptides that are associated with (i.e., attached to, bound to, etc.) the keratin nanomaterial.
Further still, the methods describing the keratin used in the manufacture of previously described keratin biomaterials yield molecular complexes that are not pure keratin nanomaterials. Even previously described “purified keratins” contain keratin complexes with tightly associated, damaged proteins, and protein fragments (i.e., peptides) that are detrimental to the self-assembly process, and/or can destabilize the biomaterial superstructure after its formation. Van Dyke M E. Wound healing compositions containing keratin biomaterials. U.S. Pat. No. 8,273,702. Sep. 25, 2012.
Keratins have been extracted from human hair fibers by oxidation or reduction using methods that are well known to those skilled in the art (see for example, Crewther, W. G., et al., The Chemistry of Keratins. Anfinsen, C. B., Jr., et al., editors. Advances in Protein Chemistry 1965, Academic Press. New York: 191-346). This chapter in Advances in Protein Chemistry contains references to more than 640 published studies on keratins and describes methods for extracting keratins. The methods described typically employ a two-step process whereby the cross-linked structure of keratins is broken down by either oxidation or reduction. If an oxidative treatment is used, the resulting keratins are referred to as keratoses and if a reductive treatment is used, the resulting keratins are referred to as kerateines. In these reactions, the disulfide bonds in cystine amino acid residues are cleaved, rendering the keratins soluble. As many of the keratins remain trapped within the protective structure of the cuticle, a second-step using a denaturing solution is typically employed to effect efficient extraction of the cortical proteins. Alternatively, in the case of reduction reactions, these steps can be combined or solutions, such as urea, thiourea, phosphates, diphosphates, sulfates, disulfates, cyanates, thiocyanates, carbonates, bicarbonates, transition metal hydroxides, surfactant solutions, and/or combinations thereof can be used (e.g., aqueous solutions of tris(hydroxymethyl)aminomethane in concentrations between 0.1 and 1.0M, and urea solutions between 0.1 and 10M).
The literature further characterizes that crude extracts of keratose and kerateines can be further refined into α-keratose, γ-keratose, acidic α-keratose, basic α-keratose, acidic γ-keratose, basic γ-keratose, α-kerateine, γ-kerateine, acidic α-kerateine, basic α-kerateine, acidic γ-kerateine, basic γ-kerateine, and keratin associated protein (KAP) fractions by a variety of methods such as isoelectric precipitation, ultrafiltration, chromatography, and combinations thereof. In a crude extract, the alpha fraction begins to precipitate below pH 6 and is essentially completely precipitated by pH 4.2. The KAP fraction generally co-precipitates with the alpha fraction, thereby producing an alpha/KAP mixture. The gamma fraction remains in solution, but can be precipitated by addition of a non-solvent. Non-solvents are water miscible but do not dissolve keratins (e.g., ethanol). Precipitation of the gamma fraction can be aided by cooling the ethanol and adding the keratin solution drop wise, rather than adding the ethanol to the keratin. Such fractionation procedures have been described in the literature and are known to those skilled in the art; however, these methods cannot yield the keratin nanomaterials described herein.
Further, many protein purification techniques are known in the art and range from fractional precipitation to immunoaffinity chromatography (for extensive treatment of this subject, see Scopes R. K. (editor). Protein purification: Principles and Practice (3rd ed. Springer, New York. 1993); Roe S., Protein purification techniques: A practical approach. (2nd ed. Oxford University Press, New York. 2001); or Hatti-Kaul R. and Mattiasson B., Isolation and purification of proteins. (Marcel Dekker AG, New York. 2003), incorporated herein by reference. For example, sub-families of acidic and basic keratins have been described by Crewther et al. as being separable by moving bounding electrophoresis, but these fractions or their properties have not been extensively described (see Crewther (1965)). Separation techniques have been applied to keratin fractions such that they can be separated into sub-fractions with useful properties, and can be re-combined into “meta keratins” with properties that are different than the starting mixtures (see Richter J. R., et al. Structure-property relationships of meta-kerateine biomaterials derived from human hair. Acta Biomater. 2012; 8(1):274-81; Van Dyke, Mark E, et al. Keratin biomaterials for treatment of ischemia. U.S. Pat. No. 8,545,893. Oct. 1, 2013; and Nunez F, et al. Vasoactive properties of keratin-derived compounds. Microcirculation. 2011; 18(8):663-9). The gelation, binding of therapeutic compounds, mechanical, and chemical properties, and other characteristics of the resulting purified keratins are established in the literature; however, the extraction and purification techniques provided are not sufficient to provide keratins without damage and/or contaminants and provide the keratin nanomaterials described herein.
A need exists for keratin nanomaterials that are capable of self-assembly into biomaterials such as hydrogels, films, foams, coatings, and fibers as well as methods for keratin nanomaterials from keratin-containing sources.
Keratin nanomaterials are macromolecular complexes existing in the form of tightly associated dimers of Type I and Type II monomers, and/or tetramers formed from tight associations of two dimers. In nature, these macromolecular complexes are unstable and quickly polymerize to form higher ordered structures. Methods described in the prior art have failed to produce stable, purified keratin nanomaterials. Keratin nanomaterials remain tightly associated in the presence of strong denaturing solutions known in the art, for example, concentrated urea solutions. Keratin nanomaterials have chemical, physical and biological properties that are distinctly different from keratinous tissues found in nature including the source hair fibers, wool, feathers and the like, as well as previously described extracted and purified keratins. For example, keratinous tissues found in nature have an inert outer layer, referred to as the cuticle in the case of hair and fur fibers. Keratin nanomaterials are not inert and can, therefore, interact with other chemical compounds, solvents, cells and cell receptors, and the like. Moreover, biomaterials prepared from keratin nanomaterials have chemical, physical and biological properties that are distinctly different from conventional keratin biomaterials described in the prior art. For example, biomaterials prepared from keratin nanomaterials form stronger network structures, owning to their high degree of self-assembly, than do conventional keratin biomaterials. This property manifests itself in the ability of keratin nanomaterials to form hydrogels at lower keratin concentration, for example. In addition, biomaterials formed from keratin nanomaterials degrade more slowly than conventional keratin biomaterials, owning to the more highly ordered molecular structure, the stability of the molecular complex, and absence of damaged peptides that can serve as catalyst to degradation. Lastly, biomaterials made from keratin nanomaterials are less immunogenic, owning to the lack of damaged peptides and the intact native structure of the molecular complex of type I and type II monomers.